Somatically hypermutated antibodies isolated from SARS-CoV-2 Delta infected patients cross-neutralize heterologous variants

SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodies—YB9-258 and YB13-292. These features are putatively introduced by somatic hypermutation and they are heavily involved in epitope recognition to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy.

strains and all samples were sorted using WT-antigens including the 3 patients infected with Delta/Gamma strains. This could potentially introduce some bias in the study and should be acknowledged as a limitation of the study.
Another concern is that the authors posits that SHM drives increased diversity and frequencies of broadly neutralizing antibodies, leading to enhanced immunities towards VOC and provided as examples, the structural mechanisms of neutralization for the 2 Mab. That repeated exposure increases SHM which increases antibody diversity is not a novel concept, however, only 24/110 (20%) binding Mab cross-reacted with other strains. Without an appropriate baseline/control group, it is hard to say that this represented enhanced immunity. The authors should test the polyclonal sera to demonstrate whether these breakthrough infection really conferred enhanced immunity. Otherwise, these B-cell clones may represent only rare subsets that do not contribute significantly to enhancing cross-reactivity.
Furthermore, while some information on the patients were given in Extended Data Table 1, I do not believe this information was sufficient to fully understand the data. Some missing information: i) Line 671 indicated that some patients only received a single dose of vaccine, however this was not indicated in Ex. Table 1 at a patient level. Furthermore, what does \ mean? ii) Blood sample was collected 7-14 days after discharge. Would be better if this was specifically indicated for each patient. It would also be useful to indicate the time between last vaccine dose and blood sampling time. iii) No vaccination or antibody titers at a polyclonal level for all patients?
Other concerns: 1. SHM is a cellular process that produces the antibodies. It is incorrect to refer to antibodies as SHMantibodies. E.g Line 73-74 and several places throughout MS. 2. Similarly, it is also incorrect to say Omicron evades neutralization by acute infection induced germline antibodies. Omicron is more resistant to neutralization by antibodies induced by strains circulating during earlier stages of pandemics even at a polyclonal level. 3. Several language issues-meaning of sentences are unclear. 4. Abstract needs to be reworked.
Reviewer #3 (Remarks to the Author): Yu and colleagues describe in this manuscript the molecular mechanism for enhanced efficacy of affinity matured neutralizing antibodies against heterologous variant of concerns (VOC). The authors analyzed memory B cells (MBC) from Delta breakthrough infections and found that they express antibodies with higher numbers of somatic hypermutations (SHMs). They isolate several antibodies able to bind the Omicron variant BA.1 and structurally analyzed two of them. Their analysis show how the variation introduced by the SMHs contribute to increase the antibody contact area with the epitope. The authors conclude that booster vaccines by increasing the evolution of SHMs in the pool of MBC, provide the immune system with resistant antibodies able to counteract the escape mutations present in the new VOC.
It has been reported in the literature that, with the emergence of VOC and the repeated antigen exposure of the population, the antibodies generated after vaccination or re-infection show affinity maturation and the increase in SHMs results in an increased binding to the spike protein (Muecksch F et al. Immunity 2021-Moriyama S et al. Immunity 2021-Sokal et al. Immunity 2021. The emergence of Omicron carrying several mutations in the RBD domain of the spike caused several breakthrough infection in the population of vaccinees. However, several authors have identified in vaccinees and infected subjects, up to 30-50% of cross-neutralizing antibodies able to bind this VOC, which are amplified by a booster vaccine eventually (Sokal et al. Immunity 2022-Lanz et al. https://doi.org/10.21203/rs.3.rs-1518378/v1 -Garcia-Beltran et al. Cell 2021-Cameroni et al. Nature 2021. Some of these antibodies in complex with the spike protein have been also structurally studied by cryo electron microscopy (Chi et al. https://doi.org/10.1038/s41392-022-00987-z -Sheward et al. https://doi.org/10.1101/2022. Structural characterization of cross-neutralizing antibodies is necessary to understand how the immune system is coping with the antigenic drift of the virus and how to design more effective vaccines. In this regard the present study is adding new knowledge to the field but it needs to be placed in the context of the published work and the obtained results need to be discussed in comparison to the mechanisms of neutralization proposed in published work. In particular some point of the paper would need further clarification: 1) Line 96: clarify the specificity of the two numbers of B cells, are these S1+ and RBD+ respectively? 2) Fig.1b: are these the RBD+ B cells? 3) Fig.1d: put the dashed lines in black; it is barely visible. Please add the mean value to these lines. It seems that there is no difference between the lines in Delta breakthrough and Non-vaccinated but the graph shows statistical significance. 4) Fig.1i: please indicate why some of the antibodies are marked in red. 5) Please add in the text (not only in the excel table) how many of the 110 selected antibodies are S1and RBD-specific and how many are only S1-specific. 6) Correct the title of Table S1; it says Table S2. 7) Line 145: Figure 2b is mentioned before figure 2a, please invert the numbering. 8) Figure 2, BLI experiment: what is S-R? Please explain. Add "wildtype" to the first row of BLI data. 9) Figure 2, neutralization graphs: please indicate that it is pseudovirus. 10) Line 146: discrepancy between binding and neutralization has been reported in other studies too. 11) Line 152: use "good" instead of "very good" especially in comparison to the binding of YB9-120 and YB13-208. 12) Line 156: There is a difference in the resistance to the RBD mutations between the two antibodies with YB9-258 showing resistance to all the mutations tested ( Fig.2c and ED Fig. 2a and b). This difference is not highlighted in the text. 13) Line 168: additional work can be cited here (Robbiani et al. Nature 2020 -Barnes et al. nature 2020). 14) Lines 173-182: please cite work reporting similar results listed in this paragraph such as: Hong et al. Nature 604, 2022-Guo et al. Cell Reports 39, 2022-McCallum et al. Science 375, 2022 15) Line 187: please explain the rationale for using the complex with two antibodies and cite previous work which used the same strategy (i.e. McCallum et al. Science 2022) 16) The binding affinity of YB9-258 and YB13-292 for the RBD of different VOC is different yet both antibodies neutralize Omicron BA.1. In light of the structural data the authors could discuss the molecular basis of this difference. Only the possible mechanism of neutralization is mentioned but not the possible interference with the binding to the different RBDs. 17) Lines 293-295: Is it possible that the high number of binding antibodies isolated by this study is the result of a bias introduced by the selection criteria adopted to identify the 270 antibodies studied? The other studies were looking at an unbiased antibody repertoire.

Reviewer #1 (Remarks to the Author):
"SARS-CoV-2 breakthrough infections induce somatically hypermutated cross-neutralizing antibodies against heterologous variants" In this work the authors aimed to understand the molecular aspects of neutralizing antibodies elicited following breakthrough delta infection to evolving SARS-CoV-2 variants of concern (VOC). The experiments utilized pooled CD19+CD27+ S1+(Delta RBD) MBC and 10x Chromium to infer V(D)J sequences. group, for WT RBD and Delta RBD, we did not observe any significant differences in plasma binding. The immunity enhancement may be weak for the breakthrough group as some of the participated patient only received one dose of vaccine prior to infection. As a result, we feel it is bit overstretching to claim "…enhanced immunity" based on our plasma binding data and we withdrew this conclusion.
2. Also, given these are elicited by previously established public clonotypes (class 1 VH3-53, class 2 VH3-21), it will be of interest to provide insights into the fraction of antibodies of public clonotype that are undergoing such SHM events.

Answer:
We have presented the average putative SHM numbers in VH genes for mAbs of known public clonotype we isolated from our patients (Fig. 1g), we found that on average they generally have more putative mutations introduced by SHM. We calculated the percentage of antibodies exhibiting above average SHM in each clonotypes and this is shown in the updated Extended Data Fig. 1c. Most VH gene families have a higher proportion of mAbs with more than 5 putative mutations, including VH3-53. However, we didn't see such trend for VH3-21. We added comments "Comparisons between the binding mAb sequences and known mAb sequences revealed that the accumulation of somatic hypermutations in isolated binding mAbs (Fig. 1g).

Answer:
We thank the reviewer to point out this interesting result. We have not yet determined the structure of YB13-281, we speculate that the binding mode of YB13-281 is different from that of YB13-292. From the structural study of YB13-292, we found that the HCDR2 insertion is important in epitope binding and likely a major contributor to the specific epitope binding mode.
Nevertheless, we highlighted differences between YB13-281 and YB13-292 in the Discussion section with an extra sentence (line 599-603 in the new tracked-change MS): "In addition to YB13-292, there is another VH3-21-encoded mAb isolated from this study, YB13-281, which displays cross-reactivity not only towards RBDs of SARS-CoV-2 variants but also SARS-CoV-1 (Fig. 1i). Different from YB13-292, YB13-281 utilizes VL1-40 for light chain and this might be responsible for its unique activity." 4. Given the ongoing evolution of SARS-CoV-2. Maybe the use of the term "broadly" neutralizing in the title may not be advisable.

Answer:
We thank the reviewer for this suggestion and we have replaced "broadly neutralizing" with "cross-neutralizing" in the title. In addition, we have changed "broadly neutralizing" to "crossneutralizing" at 4 places throughout the original text. We have also refrained from using "broadly neutralizing" in newly added text.
5. The authors should clearly mention the rate constants (kon1/2 and koff 1/2) used for comparing the apparent KD. Provided the authors used 2:1 binding model for determining the apparent KD.

Answer:
We apologize for this confusion. To briefly explain, in this study, all binding experiments were done using Protein A biosensors to immobilize IgG. We used either monomeric his-tagged RBDs or trimeric spikes as analytes to measure the interactions. Because the RBDs are monomeric, the binding sites are independent, the 1:1 binding model was able to describe the binding curves, and for all the binding experiments with RBDs, only one kon, koff and KD were reported (these are summarized in Extended Data Tables 3, 4 and 6). For binding experiments with spikes, likely due to multivalent binding, we found 2:1 model was required to fit the binding curves properly, hence we used the 2:1 binding model and reported kon1, kon2, koff1, koff2 and associated KDs (reported in Extended Data Table 2).
For comparison of KD values, we only used RBD binding data, so only one KD from each binding curve (derived from koff/kon) was involved in comparison. We have also included more details "Data were reference-subtracted and analyzed using Data to binding models used for spike and RBD data respectively to clarify the data analysis procedure.
6. The local resolution and map fits needs to be provided at the binding interface. To provide clarity of the model to map.

Answer:
We thank the reviewer for the suggestion. We updated Extended Data Fig. 4d and Extended Data Fig. 4i to include local resolution assessments zoomed in at the antibody-antigen interfaces.

Reviewer #2 (Remarks to the Author):
The manuscript by Yu et al., describes the isolation and characterization of monoclonal antibodies from vaccinated and unvaccinated patients that were infected with the SARS-CoV-2 Delta variant. The noteworthy results were the structural characterization and modeling of two of the most potent mAB, YB9-258 and YB13-292. YB9-258 presumably showed highly mutated residues while YB13-292 showed an insertion of SNIL motif into the antibody. Whilst these findings are novel and useful to the field, the manuscript is weakened by some overinterpretation of data in the first part of the manuscript.

Answer:
We thank the reviewer to point out the novelty and limitations of the study. We appreciate the constructive suggestions offered for manuscript improvement. We made detailed modifications after studying the reviewer's comments. We thank the reviewer to point out limitations in this study. We would like to explain the use of pooled sample first. As the designated hospital for COVID-19 treatment in the Guangzhou area, we are responsible exclusively for treatment of all COVID-19 patients from cities of Guangzhou, Foshan and Dongguan. Based on understanding of helping better understanding of COVID-19, some patients kindly agreed to donate blood for research, however, they would normally decline further sample collection after quarantine (they were quarantined after hospital discharge) as it would be too much of a bother for their daily lives. Therefore, although we could recruit many patients, their blood samples are very precious. In addition, our primary aim was to isolate neutralising antibodies. Our preliminary experiment showed that under the setup we have, we encountered low cell sorting efficiency for certain samples. In order to increase the number of recovered cells for single cell sequencing and to maximise SARS-CoV-2 specific B cell recovery, we pooled the samples. Lastly, according to previous experience in our laboratory, single-cell transcriptome sequencing with fresh blood has been more effective, we had to pool blood samples collected in the same day on which the patients agreed to donate blood for the last time before their quarantine ended to allow samples to be processed on the same day. By analysing single cell sequencing data derived from the samples prepared this way, we were able to isolate a few cross-neutralising antibodies. We do apologise for confusions may be caused by sample mixing and in the updated MS we attempt to describe the overall characteristics of the single cell sequencing data while try to minimize over-interpretation of the data following the suggestions offered by the reviewer.
After studying the reviewer's careful review report, we found inaccuracy in the patient sample information, therefore we want to address reviewer's point i) first.
Furthermore, while some information on the patients were given in Extended Data Table 1, I do not believe this information was sufficient to fully understand the data. Some missing information: i) Line 671 indicated that some patients only received a single dose of vaccine, however this was not indicated in Ex.

Answer:
We thank the reviewer's careful review of the manuscript. In the original table, the "\" means "vaccine status not confirmed". We apologise for including these samples and confusion caused.
Following the reviewer's suggestion, we carefully reviewed patient information, and we found several patients with immunization status that needs further confirmation, we removed groups with such patients (but sequencing data derived from the removed groups were deposited together with the retained groups: https://ngdc.cncb.ac.cn/gsa-human/s/ASvvWkms). As a result, we only retained groups YB9, YB12, YB13 and YB14. We also removed groups in our non-vaccinated group who were infected with Delta and Gamma variants following the reviewer's suggestion. The new table now contains more information of individual patient in each group including gender, age, infected virus strain, disease severity, symptoms, vaccine type, vaccine dose, duration of hospitalization, sample collection time, and individual IgG titres for easier understanding of the data. As a result of the above stated change, we reanalysed the data, we updated Fig. 1 and Extended Data Fig. 1 with the new analysis. Within the retained groups, two patients unfortunately confirmed that they were not vaccinated, so we changed descriptions of the group in several places as "group primarily with Delta-breakthrough infections" or "Delta-infected group" instead to be accurate. We present a table below to summarise the changes to the analysis results.     Fig. 1d and Extended Data Fig. 1b. We also removed the non-vaccinated groups infected with Delta/Gamma strains to minimize potential bias.

clusters. ……d, Density plot showing SHM counts on variable heavy-chain gene (VH) sequences of B cells from Delta-infected patients, non-vaccinated patients and antibodies from
After removal of the non-vaccinated groups with Delta/Gamma strain infections, we found that there was no significant difference on the average mutation numbers of VH genes expressed by B cells between the two patient groups (shown in the updated Fig. 1d). However, we did observe a higher proportion of B cells with unmutated VH genes in non-vaccinated group (added as a new panel in the updated Fig. 1d).
Also based on this analysis, the following changes were made: We also updated relevant sections in Fig. 1 legend for panels d and   We clearly state in the text that the analysis results represent "on average" for the population we isolated the antibodies from.
We also added a "Limitation of the study" section.  Supplementary Fig. 1b. We divided the plasmas into non-vaccinated infected groups and a group with Delta-variant breakthrough infections (details for each patient in pooled groups are shown in updated Extended Data Table 1, binding data for the 2 patients without vaccination in pooled sample groups YB9 and YB12 are shown within the breakthrough group but not included in statistics analysis, therefore the group is an authentic breakthrough group). The trend of decreasing binding towards succeeding mutant RBDs is obvious within the groups. Although there is some evidence to show that the breakthrough infected group has higher binding (*p<0.05) towards Omicron RBD than the non-vaccinated group, for WT RBD and Delta RBD, we did not observe any significant differences in plasma binding. The immunity enhancement may be weak for the breakthrough group as some of the participated patient only received one dose of vaccine prior to infection.
Therefore, we agree with the reviewer it is over-stretching to claim "enhanced immunity". We ii) Blood sample was collected 7-14 days after discharge. Would be better if this was specifically indicated for each patient. It would also be useful to indicate the time between last vaccine dose and blood sampling time.

Answer:
We thank the reviewer for this suggestion. We have included "Sample collection time" for each patient in Extended Data Table 1 We would also like to clarify the sample collection time, people with COVID-19 were sent to Guangzhou 8 th people's hospital, the designated hospital for COVID-19 treatment in Guangzhou area. Once the patient was tested negative for virus RNA, he or she was discharged from the hospital treatment (this is the discharge time referred in the Extended Data Table 1) but was further quarantined for 14 days. Convalescent blood samples were collected during this quarantine period.

iii) No vaccination or antibody titers at a polyclonal level for all patients?
Answer: We thank the reviewer for this suggestion. We now included vaccination information and antibody titer for each patient in the updated Extended Data Table 1, we also visualized the antibody titer data at a polyclonal level in Supplementary Fig. 1a. The antibody titer for all samples were tested for SARS-CoV-2 (WT) specific antibody using a commercially available kit. We included the method for antibody titer detection in the Methods section. (Line 1342-1352 in the new tracked-change MS) Other concerns: 1. SHM is a cellular process that produces the antibodies. It is incorrect to refer to antibodies as SHM-antibodies. E.g., Line 73-74 and several places throughout MS.

Answer:
We thank the reviewer for this suggestion, we updated almost all sentences about "SHM" in our manuscript and we regard SHM as a cellular process. These modifications include:

Answer:
We thank the reviewer for this suggestion, we replaced the sentence as "with extraordinary abilities in evading antibodies isolated earlier in the pandemic" deleting words "acute-infection induced germline" in the Abstract.
3. Several language issues-meaning of sentences are unclear.

Answer:
We updated the manuscript according to the PDF kindly provided by the reviewer. These corrections are listed in the last part of this section.
4. Abstract needs to be reworked.

Answer:
We updated the abstract according to suggestions from the reviewers.

Revisions according to the annotated PDF file kindly provided by the reviewer:
Line 1 increased values leading to a greater number of clusters. It is reported in the Seurat guided tutorial that resolution between 0.4-1.2 typically returns good results for single-cell datasets of around 3000 cells (https://satijalab.org/seurat/articles/pbmc3k_tutorial.html). Therefore, we set a resolution of 1 to ensure that more clusters were identified. After clustering, we annotated the cells using SingleR, an automatic annotation method for single-cell RNA sequencing data. It is also used in many studies (Cao et al, Cell, 2020;Huuhtanen et al, Nat Commun, 2022;Mair et al, Nature, 2022). Labels of cells were determined by their similarity to the reference dataset with known labels. We also manually modified the labels according to the expression of classic marker genes such as IGHC genes.
We added more details in the "Cell-type annotation and differential expression analysis" part in the Methods section:  (Macosko et al, Cell, 2015).

Answer:
In the original manuscript, we simply pointed out a few VH gene families with binding ratios higher than 50%, we did not mention the 4-4, 1-21 VH gene families here because the number of binding antibodies isolated for these 2 families was low. In the updated manuscript, we used the following criteria to define the VH gene families with high binding ratios: (1) VH gene families containing more than 5 mAbs; (2) proportion of binding mAbs higher than 60%. The list of VH gene families was changed from "IGHV1-46, IGHV3-21, and IGHV3-66" to "IGHV2-5 (100.00%),

Answer:
We apologize for the confusion, here we were trying to corelate our SHM levels of isolated antibody sequences (Fig. 1g of previous MS) with the "on-average" SHM level of the VDJ transcriptome data (Fig. 1d of previous MS). We revised the sentence as: (Revised) "Comparisons between the binding mAb sequences and known mAb sequences revealed the accumulation of somatic hypermutations in isolated binding mAbs (Fig. 1g). This is consistent

to our observation that B cells from patients primarily with Delta variant breakthrough infections
show sign of increased level of somatic hypermutation by single cell V(D)J transcript data (Fig.   1d)." (Line 272-276 of the new tracked-change MS).
Line 146: We corrected the spelling of "correlating". (Line 339 of the new tracked-change MS).
Line 148: We have changed "compromised but substantial" to "reduced but still substantial" (Line 341 of the new tracked-change MS) Line 290: "No discussion of limitation?" We added a "Limitation of the study" section at the end of the main text in the new manuscript.
(Line 654-662 of the new tracked-change MS).
Line 293: "I am not convinced that "many" binding antibodies are able to cross-react to Omicron. If I read the results correctly, the authors stated 14/110 isolated could bind to Omicron in a BINDING assay, and only 6 bound Omicron spike?" Answer: We changed "many" into "some". for pseudovirus neutralization below 1 μg/mL (Supplementary Tables 2 and 3). Although the percentages in these two studies are close, the 247 neutralizing antibodies reported by groups worldwide may be biased towards more potent ones among others in the reported antibody isolation exercises. Therefore, the actual percentage of previously isolated antibodies which still retain binding or neutralizing to Omicron BA.1 may be lower.
We added comments: "Previously, it has been identified that only 32 of the 247 previously Line 482: "Y-axes in Figure 2c YB13-292 is off by a log? Why is wild type at 1000?" Answer: The KD comparisons used KD values cited in Extended Data Table 3. The relative binding affinity to WT RBD by YB-258 and YB13-292 had been arbitrarily defined as 100 and 1000, this was done to facilitate easier comparison within their respective groups so that relative KD values in each group would have integer values. To remove confusion, we have normalized the relative binding affinity to WT RBD to 1 instead in Fig. 2c and Extended Data Fig. 10d. We updated Fig. 2c and Extended Data Fig. 10d.
Line 666: "Data missing. Please provide further clarification on whether subjects received 1 or 2 doses, and at least a summary statistic of when the last dose was administered.
Unvaccinated-what kinds of symptom severity?
Also, is there data on vaccine and delta titers for the subjects?" Answer: We included more comprehensive patient information, including vaccine doses, symptoms, disease severity, etc. in Extended Data Table 1. Unfortunately, we have no data about the time of the last vaccine dose.
Line 675: "average duration of sample collection between vaccinated and non-vaccinated groups? Long hospitalization due to prolonged shedding can occur" Answer: We added hospitalization durations for patients in the Extended Data Table 1.
Line 886:"what does slash mean?? single or two doses?"

Answer:
The slash means "no information" in the old MS. We updated the information of vaccine and dose in Extended Data Table 1.

Reviewer #3 (Remarks to the Author):
Yu and colleagues describe in this manuscript the molecular mechanism for enhanced efficacy of affinity matured In this regard the present study is adding new knowledge to the field but it needs to be placed in the context of the published work and the obtained results need to be discussed in comparison to the mechanisms of neutralization proposed in published work.
We thank the reviewer for recognizing new information provided by this study and pointing out specific directions for manuscript improvement. We have added most of the references the reviewer had mentioned in appropriate places in the discussion section of the new manuscript.
(Line 610-618 of the new tracked-change MS).
In particular some point of the paper would need further clarification: 1) Line 96: clarify the specificity of the two numbers of B cells, are these S1+ and RBD+ respectively?

Answer:
We apologize for the confusion.
From the patient PBMC, CD19 + B cells were first enriched from pooled PBMCs with Miltenyi CD19 MicroBeads kit. The enriched CD19 + B cells were then stained with PE labelled antihuman CD27 antibody, SARS-CoV-2 biotinylated RBD protein (His Tag) conjugated with FITCstreptavidin, and biotinylated S1 protein (His Tag) conjugated with APC-streptavidin.
The purity of sorted cells was rechecked by flow-cytometry, we did not verify the antigen specificity of the sorted B cells with other methods. So, the sorted cells were denoted as CD19 + CD27 + Delta-RBD + Delta-S1 + specific memory B cells. We have added more details in the Methods section for clarification.
Previously described at Line 96 of the original manuscript, after removal of several patient groups, the number of single-cell transcriptome data and single-cell V(D)J data are for 3286 and 3554 B cells respectively. The reason for the B cell number difference is that for certain cell we were not able to obtain both the single-cell transcriptome data and single-cell V(D)J data after respective quality controls. We have added the following sentence in the section describing our sequencing data to further explain how the sequencing data were obtained.

Answer:
As explained above, these are CD19 + CD27 + Delta-RBD + Delta-S1 + B cells. We also explicitly stated which cell sequencing data were used for 3) Fig.1d: put the dashed lines in black; it is barely visible. Please add the mean value to these lines. It seems that there is no difference between the lines in Delta breakthrough and non-vaccinated but the graph shows statistical significance.

Answer:
We thank the reviewer's suggestion. We updated Fig. 1d with the dashed lines changed to black and the mean values marked. After removing several pooled-samples, we found that there was no significant difference on average SHM between these two groups. However, we observed a significantly higher proportion of unmutated VH sequences for B cells from nonvaccinated group, showing elevated SHM level in the patient group primarily with Delta variant breakthrough infections. We presented the new analysis results in the updated Fig. 1d. Fig.1i: please indicate why some of the antibodies are marked in red.

Answer:
The six antibodies were more potent cross-neutralizing antibodies and we therefore analysed them in detail and coloured them in red in the previous MS. We have added this information to the legend of Fig. 1i: "Six potent cross-neutralizing mAbs (IC50 < 0.05 mg/ml) were marked with *." (Line 855-856 of the new tracked-change MS).

5)
Please add in the text (not only in the excel table) how many of the 110 selected antibodies are S1-and RBD-specific and how many are only S1-specific.

Answer:
We tested all the 117 antibodies by RBD binding and 53 are only RBD-specific, we selected some of the tighter RBD binder and further tested their S1 binding, we found 34 of the 53 selected antibodies are S1-and RBD-bispecific. Because we did not test all antibodies for S1 binding, so we do not want state their S1 specificities. Table S1; it says Table S2.

Answer:
We revised the information and updated Supplementary Tables 1-3.

Answer:
We inverted the sequence of Fig. 2a and b. 8) Figure 2, BLI experiment: what is S-R? Please explain. Add "wildtype" to the first row of BLI data.

Answer:
S-R is a construct where we mutated the multibasic S1/S2 cleavage site PRRAR to a single R.
Detailed structure characterization was performed for this construct and compared to the 2P construct in Xiong X. et. al. NSMB, 2020 (DOI: 10.1038/s41594-020-0478-5), and the data showed that S-R spike has more closed spikes. Probably due to this property S-R spike somewhat reduces binding of various type of antibodies comparing to 2P spike (He P. et. al. Nature Microbiology, 2022, DOI:10.1038. Following the reviewer's suggestion, we have changed the "S-R" designation as "S-R/Wildtype". For clarity, we also added more information in the "Protein expression and purification" section of Methods. (Line 1386-1399 of the new tracked-change MS). Figure 2, neutralization graphs: please indicate that it is pseudovirus.

Answer:
We added "pseudovirus neutralization" in the Fig. 2a panel title.
10) Line 146: discrepancy between binding and neutralization has been reported in other studies too.

Answer:
We thank the reviewer for this suggestion. We added relevant references to the manuscript and revised the sentence as: "It has been reported in other studies that binding activities are not always correlating with antibody virus neutralizing activities (Yuan M, et al. Science. 2020, Wec AZ, et al. Science 2020." (Line 343-344 of the new tracked-change MS).
11) Line 152: use "good" instead of "very good" especially in comparison to the binding of YB9-120 and YB13-208.

Answer:
We have changed "very good" to "good". (Line 346 of the new tracked-change MS).
12) Line 156: There is a difference in the resistance to the RBD mutations between the two antibodies with YB9-258 showing resistance to all the mutations tested ( Fig.2c and ED Fig. 2a and b). This difference is not highlighted in the text.

Answer:
We thank the reviewer for this comment. We highlight the difference by adding the sentence  (Fig. 2c and Extended Data Fig. 2a).
While YB13-292 is resistant to most single RBD mutations that were previously known to be highly detrimental to binding of many known RBD antibodies (Harvey, W. T. et al. Nat Rev Microbiol 2021) (Fig. 2c and Extended Data Fig. 2b). RBD  The rationale was complex we were originally intended to obtain complex with higher molecular weight near the epitope to allow EM software to do focus refinement to improve resolution.
However, we found simultaneous incubation with antibodies YB9-258 and R1-32 disrupted the spike and the S1 fragment in complex with 2 Fab was suitable to facilitate high resolution structure determination near the YB9-258 binding interface. We added "In order to obtain more suitable samples for high resolution cryo-EM structure determination, after extensive efforts, following a similar strategy as previously described (McCallum et al. Science 2022)," (Line 413-415 of the new tracked-change MS).
16) The binding affinity of YB9-258 and YB13-292 for the RBD of different VOC is different yet both antibodies neutralize Omicron BA.1.
In light of the structural data the authors could discuss the molecular basis of this difference. Only the possible mechanism of neutralization is mentioned but not the possible interference with the binding to the different RBDs.

Answer:
We thank the reviewer for this suggestion. We added the following comments in sections describing structures of YB9-258 and YB13-292 epitopes respectively: Line 429-435 of the new tracked-change MS: "In this epitope, residue 417 is centrally located while residues 484, 452 and 490 are outside of the epitope. K417N has been usually found to completely abolish binding of "class 1" VH3-53 antibodies (Yuan, M., et. al., Science 2021, Wang, R., et al., Immunity 2021. Although K417N only mildly affects YB9-258 binding but its effect is the strongest among common RBD single substitutions we tested (Fig. 2c, left panel).  (Figs. 2c and 4b).
Substitutions at 417 and 478, outside of the epitope, have very little effect (Figs. 2c and 4b).
However, different from 47D1, for which E484K and F490S were able to completely abolish binding, YB13-292 is able to maintain reasonable binding to RBDs with E484K and F490S substitutions (Extended Data Fig. 2b, c)." 17) Lines 293-295: Is it possible that the high number of binding antibodies isolated by this study is the result of a bias introduced by the selection criteria adopted to identify the 270 antibodies studied? The other studies were looking at an unbiased antibody repertoire.

Answer:
In fact, we found (with the latest analysis) "13/117 (~11%) Delta binding mAbs can bind to Omicron BA.1 RBD", this percentage may be quite high. Therefore, our selection criteria we adapted from Cao's method (Cao, Y., et al., Cell, 2020), may be indeed biased towards identifying cross-reactive antibodies. Answer: We apologize for the unclarity, the masks were represented in the Extended Data Fig. 3 but were not very obvious. We have increased the contrast of the yellow color for the masks and we high-lighted the intermediates structures with yellow boxes on which yellow masks were applied for local density extraction.

OTHER CHANGES
We updated "Omicron" in the original manuscript to "Omicron BA.1" throughout the manuscript.
For antibodies, YB9-120, we investigated their binding to Omicron BA.2, BA.3 and BA.4 spikes and RBDs and these data are included in Fig, 2b and Extended Data Fig. 11.
We have deposited the single cell sequencing data (including those for the groups mentioned in the original manuscript, with the accession number PRJCA012020) in the National Genomics Data Center (https://bigd.big.ac.cn/), it will be a validated by the curator and it should be available upon publication, the preview link is included (https://ngdc.cncb.ac.cn/gsahuman/s/ASvvWkms). Cryo-EM density maps and structural models have been deposited in the EMDB and PDB, the accession numbers are summarized in the structural data statistics  Table 3